Mechanistic studies of the adjuvant effects of CTA1-DD and the native cholera toxin:

Mechanistic studies of the adjuvant effects of CTA1-DD and the native cholera toxin:

Impact of cell targeting and tissue localization

Johan Mattsson

Department of Microbiology and Immunology Institute of Biomedicine

University of Gothenburg

Sweden, 2012

Mechanistic studies of the adjuvant effects of CTA1-DD and the native cholera toxin:

Impact of cell targeting and tissue localization

Johan Mattsson

Department of Microbiology and Immunology, Institute of Biomedicine, University of Gothenburg, Sweden, 2012.

Abstract

Vaccines are the most effective means of preventing infectious diseases and improving global health. However, few vaccines have successfully been developed for protection at mucosal surfaces where most pathogens gain access. The reason for this poor outcome has been the lack of immunoenhancers, or adjuvants, that allow for efficient mucosal immunizations. Empirical data has identified cholera toxin (CT) as one of the most effective adjuvant molecules known today. Because of its inherent toxicity, clinical use of CT is precluded. The closely related CTA1-DD adjuvant share the same dependence on the ADP-ribosylating enzymatic activity of the A1 subunit, however the differential binding properties of CTA1-DD renders the molecule safe and non-toxic. The aim of this thesis work has been to increase the knowledge about how adjuvants function by studying CT and the CTA1-DD adjuvant. To delineate key elements required for the adjuvant effects, we explored their in vivo distribution in tissues and the dependence on specific components of the immune system.

We found that both CT and CTA1-DD localizes to the marginal zone macrophages (MZMs) of the spleen after iv. injection. To investigate the importance of this finding we treated mice with clodronate liposomes, depleting the MZMs, and found that immunizations with CT or CTA1-DD generated unperturbed immune responses in the treated mice, suggesting that this cell subset is dispensable for their adjuvant effect.

Following initial accumulation in MZMs, CTA1-DD localized to the follicular dendritic cell (FDC) network.

This correlated with the ability of CTA1-DD to activate complement primarily via the alternative pathway, allowing the adjuvant to bind to the complement receptors 1 and 2 (CR1/CR2) on FDCs. We found that adjuvanticity was dramatically reduced in Cr2 knockout mice, where this localization is absent. This prompted us to isolate FDCs from mice immunized with CTA1-DD and assess their activation status using RT-PCR. We found that a number of genes important for the ability of FDCs to support germinal center (GC) formation were up-regulated. Whereas FDCs are highly involved in orchestrating the GC reaction it was feasible that a direct effect of CTA1-DD on FDC functions promoted GC formations.

Conventional dendritic cells (DCs) are believed to be essential for generating follicular helper T (Tfh) cells, but it is unknown to what extent CTA1-DD affects this process. Unexpectedly, when using the CD11c-DTR mouse model to deplete DCs, we found Tfh cell priming appeared to be normal in terms of expansion and phenotype, however a significant reduction in the expression of the Tfh cell transcription factor Bcl-6 was recorded following immunization. Despite potentially reduced Tfh function, we observed that the ability of CTA1-DD to promote antibody production and GC formations was still significant. We speculate that this was the result of a compensatory mechanism employed by the CTA1-DD adjuvant, possibly via the activation of FDCs.

Finally, we examined the immunomodulatory properties of CT. CT is generally considered a Th2 adjuvant and has been reported to inhibit Th1 responses by down-regulating IL-12 production. Here we demonstrated that CT rather induces a mixed Th1/Th2/Th17 response, independently of IL-12. Interestingly, i.v immunization with CT completely blocked the ability to respond to a subsequent immunization, and both Th1 and Th2 responses were inhibited, arguing that an early event in the priming process was impaired. This correlated well with the observation that CD11b⁺ DCs were activated, thus compromising their ability to process additional antigens. In addition we found that the CD8α⁺ DC population was depleted following CT-administration and could therefore not be involved in the adjuvant effect of CT. Finally, reconstituting CT-treated mice with DCs re-established their ability to respond to a subsequent immunization.

In conclusion, we have demonstrated that the differential binding properties of the related adjuvants, CT and CTA1-DD, critically affects the mechanisms by which they modulate immune responses. This underpins the importance of targeting adjuvants to specific components of the immune system in order to efficiently deliver stimulation and avoiding toxic side effects, an important insight when designing future vaccines.

Keywords: adjuvants, vaccines, CTA1-DD, cholera toxin, follicular dendritic cells, dendritic cells, Th1, Th2, Th17, Tfh, germinal centers, complement.

ISBN: 978-91-628-8492-5

Original papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals (I-III):

I. Complement activation and complement receptors on follicular dendritic cells are critical for the function of a targeted adjuvant

Mattsson J, Yrlid U, Stensson A, Schön K, Karlsson MC, Ravetch JV, Lycke NY.

J Immunol. 2011 Oct 1;187(7):3641-52

II. The adjuvant function of cholera toxin is independent of IL-12 and mediated by CD11b

CD11c

dendritic cells inducing not only Th2- but also Th1 and Th17 responses

Mattsson J, Schön K, Yrlid U, Lycke NY.

Manuscript

III. CTA1-DD adjuvant targets follicular dendritic cells and up-regulates the expression of germinal center-promoting genes

Mattsson J, Gustafsson T, Dahlgren M, Stensson A, Johansson-Lindbom B, Yrlid U, Lycke NY.

Manuscript

Reprints were made with permission from the publisher

7

Table of contents

Abstract

Original papers Table of contents

Abbreviations 8

Introduction 11

Vaccines 12

Adjuvants 14

Aluminum salts 14

Squalene based formulations; MF59 and ASO3 15

Monophosphoryl lipid A 16

Cholera toxin 16

CTA1-DD 18

The complement system 19

The cell types and the microanatomy of the spleen 21

Marginal zone B cells 22

Macrophages of the marginal zone 23

Dendritic cells 23

T cell differentiation 25

Th1 25

Th2 26

Th17 27

T regulatory cells 27

Additional Th subsets 27

Germinal centers 28

Somatic hypermutation 29

Class switch recombination 29

Tfh 30

Long lived plasma cells and memory B cells 31

Follicular dendritic cells 31

Aims

Key methodologies

Mice and immunizations 36

Immunohistochemistry 36

Antibody assay 37

Preparation of fusion protein and CT conjugates 38

Cell sorting and quantitative real-time PCR 38

Bone marrow chimeras 39

Results

CTA1-DD localizes to follicular dendritic cells in a complement dependent manner 40

CTA1-DD is dependent on complement for full adjuvant function 40

The expression of complement receptors on FDCs mediates the adjuvant function of CTA1-DD 42

The role of classical DCs in the CTA1-DD adjuvant effect 43

CTA1-DD directly activates FDCs 45

Marginal zone macrophages are essential for accumulating CT in the marginal zone but

dispensable for the induction of immune responses 46

CT immunization stimulates the generation of a mixed Th1/Th2/Th17 response 46

CT preferentially activates CD11b⁺ DCs 47

CT pre-treatment abolishes the response to subsequent immunization 48

Discussion

Acknowledgements

References

Papers I-III

8

Abbreviations

ADP Adenosine diphosphate APC Antigen presenting cell

ARF Adenosine diphosphate ribosylating factor Baff B lymphocyte activating factor

BCR B cell receptor C4BP C4 binding protein

CR1/2 Complement receptor 1/2 CSR Class switch recombination

CT Cholera toxin

CTB Cholera toxin subunit B CTL Cytotoxic lymphocyte

DAMP Damage-associated molecular patterns

DC Dendritic cell

DTR Diphteria toxin receptor DTx Diphteria toxin

DZ Dark zone

ER Endoplasmatic reticulum

ERAD ER associated protein degradation

FcR Fc receptor

FDC Follicular dendritic cell FOB Follicular B cell

GC Germinal center

GFP Green fluorescent protein

HPRT Hypoxanthine guanine phosphoribosyltransferase

IFN Interferon

IL Interleukin

in Intranasal

iTreg Inducible T regulatory cell

iv Intravenous

LPS Lipopolysaccharide LT Heat labile enterotoxin

LTB Heat labile enterotoxin subunit B

LZ Light zone

MAC Membrane attack complex

MARCO Macrophage receptor with collagenous structure MBL Mannose binding lectin

MHCII Major histocompability complex class II MMM Metallophilic macrophages

MPLA Monophosphoryl lipid A

MZ Marginal zone

MZB Marginal zone B cells MZM Marginal zone macrophages NLRP3 NOD-like receptor protein 3

NP-CGG (4-hydroxy-3-nitrophenyl)acetyl (NP)-chicken γ-globulin (CGG) nTreg Natural T regulatory cells

OVA Ovalbumin

PC Plasma cell

PRR Pattern recognition receptor

9

RA Retinoic acid

SHM Somatic hypermutation S1P Sphingosine 1-phosphate SRA Scavenger receptor A

STAT Signal transducer and activator of transcription TI T cell independent

TCR T cell receptor

TD T cell dependent

Tfh T follicular helper

Th T helper

TLR Toll like receptor TNFα Tumor necrosis factor α

WT Wild type

10

11

Introduction

During the 20

century, the average life expectancy has dramatically increased worldwide, from an average life span of only about 30 years in the early 1900 to almost 65 years at the turn of the century [1]. The principal reason for this remarkable progress has been the control of infectious diseases by the invention of antibiotics and vaccines [2]. Vaccines are unique in that they provide prophylactic protection against diseases, many of which lack effective therapeutic treatment. Up until recently, vaccine development was largely a process of trial and error using formulations based on attenuated or killed pathogens, often mixed with the adjuvant alum. This strategy has worked remarkably well in the past, many of these vaccines have been highly successful. However some diseases such as HIV or malaria represent a challenge that will require a new approach to vaccine development. In order to be successful, these new vaccines will require rationally selected conserved antigens that, in contrast to whole cell vaccines, are safer and will be able to confer protection against infections that fail to generate natural immunity. In addition, such vaccines will require new types of immunoenhancers, or adjuvants, that can modulate the immune response not only by enhancing the effect of a vaccine, but also by influencing the quality of the response.

Furthermore, in addition to systemic immunity, protection against many infections require local mucosal defenses. In order to avoid inappropriate immune responses against harmless environmental antigens, mucosal surfaces are biased towards the development of tolerance.

Therefore, powerful adjuvants are needed in order to boost the efficiency of vaccines delivered by mucosal routes.

The mechanisms behind the immunostimulatory effect of adjuvants used in human vaccines are still not fully understood. For example, the by far most widely used adjuvant, alum, has been included in vaccines for over 80 years, nevertheless its impact on the immune system remain unclear. In order to develop new adjuvants that will be included in future vaccines, it is critical to increase the knowledge about how existing adjuvants modulate the immune response.

The aim of my thesis has been to explore the immunostimulatory mechanisms behind two

related adjuvants; cholera toxin (CT) and CTA1-DD. We have investigated the targeting

properties of the two adjuvants as well as their dependence on selected cell types- and other

components of the immune system. For example the ability of CTA1-DD to promote the

formation of large and numerous germinal centers (GC), crucial in the development of high

quality memory B cells, was correlated to the activation of complement and the binding of the

adjuvant to follicular dendritic cells (FDC). We also investigated the importance of

conventional dendritic cells (DC) and their role in the differentiation of T follicular helper

cells in this process. Despite the fact that the key factor behind the adjuvant function of both

CTA1-DD and CT is the enzymatic activity of the shared A-subunit, their effect following

immunization is fundamentally different. While CT is highly toxic and paradoxically can

potentiate immune responses as well as induce a state of hyporesponsiveness following

administration, CTA1-DD is non-toxic and stimulates immune responses without exhibiting

12

the immunosuppressive effect as seen with CT. Thus, a fundamental question that we address is whether CT and CTA1-DD promote immune responses by acting on the same target cells or if they employ different cell types and mechanisms for their immunoenhancing effects.

Furthermore we challenge previous reports describing CT as an adjuvant that primarily promotes Th2- and not Th1 responses. In contrast we demonstrate a balanced induction of Th1, Th2 and Th17 immune responses following immunization with CT. In the following sections I will describe key aspects of adjuvants and the immune system relevant to this thesis work.

Vaccines

The basis for vaccination was set at the end of the 18

century by Doctor Edward Jenner. At this time it was widely believed that dairymaids, who often contracted cowpox, were also protected against smallpox. In a classical experiment, Jenner inoculated an 8 year old boy with matter from a cowpox lesion derived from the hands of a young dairymaid. The boy developed mild fever but soon recovered. When Jenner inoculated the boy again, this time with matter from a fresh smallpox lesion, no disease developed. Jenner concluded that the boy was protected [3]. However, Jenner was not the first to induce immunity by inoculation. A practice where non-immune individuals were inoculated with smallpox intradermally, referred to as variolation, had been used long before Jenner’s experiments. But, variolation was risky, a significant number of inoculated persons developed the disease and the risk of contracting other infections was considerable [4]. The importance of Jenner’s discovery, which remains to be fundamental in the field of vaccinology, was that it is possible to infer protection against a severe disease using a similar agent that cause mild- or no symptoms. The initial work of Jenner and his successors resulted in the eradication of smallpox in 1977. Today, vaccination is recognized as one of the greatest public heath achievements of the 20

century. The eradication of smallpox was followed by an almost complete elimination of polio and a major reduction in the prevalence of a number of diseases including diphtheria, tetanus, pertussis, yellow fever, Haemophilus influenzae type B, measles, mumps, rubella, typhoid fever and rabies [5].

Vaccines can be broadly divided into live attenuated and nonliving vaccines. The live attenuated vaccines, such as the smallpox vaccine, are comprised of weakened versions of the pathogen, closely mimicking the natural infection, but with mild or no symptoms. Such vaccines are highly efficient and confer long lasting protection because they provide immunological memory typified by an ability to mount a strong response within a few days.

However there are apparent risks with attenuated vaccines as they can cause severe infections,

particularly in immunocompromised individuals. There is also a risk that the attenuated

organism can revert to a highly virulent pathogen [6]. Hence, live attenuated vaccines are

unsuitable when dealing with pathogens that mutate rapidly, such as HIV, or for those that

exist in many different serotypes, e.g. dengue fever. Of course, attenuated vaccines against

infections that naturally provide no- or only partial protection against reinfection are unlikely

to be successful [7].

13 Nonliving vaccines can be further classified as whole cell - or subunit vaccines. Such vaccines provide a better safety profile as compared to attenuated vaccines. As the name implies, whole cell vaccines contain the killed pathogen that has been inactivated using chemicals, high temperatures or radiation. Subunit vaccines on the other hand are comprised of one or several antigens purified from the pathogen or produced recombinantly. The subunit approach offers several advantages in addition to their improved safety features. They can be designed to protect against multiple serotypes of a pathogen by including a variety of antigens in the vaccine formulation or by directing the response towards conserved epitopes present on many strains. However they are also less immunogenic and most often require adjuvants to generate a strong immune response.

The majority of licensed vaccines available on the market today are administered via parenteral routes, either intramuscularly or subcutaneously. Although efficient at generating systemic immunity, such vaccines are often poor at inducing mucosal responses [8, 9].

Mucosal surfaces represent the primary entry point for a large number of pathogens, therefore mucosal immunity is much warranted to prevent mucosal as well as systemic infections.

Furthermore, mucosal vaccines are needle free, eliminating the risk of spreading infections by contaminating pathogens and they most often imply improved compliance, especially in children and in individuals that suffer from a fear of needles.

Despite the advantages of mucosal immunization there are only 7 licensed mucosal vaccines available, 6 delivered by the oral route and one intranasal (in), all of which are live attenuated or whole dead formulations [10]. This reflects the inherent challenges of delivering antigen- and initiating immune responses at mucosal surfaces. A mucosal vaccine candidate must be able to penetrate mucosal barriers such as the mucus layers or epithelial cells. In addition it must be protected from degradation so that it can reach the immune inductive sites in the mucosal immune system. The degradation of antigens is primarily an issue associated with oral vaccines, due to the local enzyme enriched environment in the gut. To overcome this problem oral vaccines use large quantities of antigen and often include buffers to counter the acidity of the stomach. Notable disadvantage with oral vaccines is their varying efficiency as well as their often poor induction of memory responses [11]. In. vaccination offers an advantage over oral vaccines in that lower amounts of antigen are required and that both mucosal and systemic protection is effectively generated [12, 13].

A major challenge in the development of mucosal vaccines is to overcome the largely

tolerogenic environment of mucosal surfaces. In order to prevent immune responses towards

environmental antigens or commensal bacteria, mucosal surfaces generally induce tolerance

as opposed to immunity. The tolerogenic property of mucosal surfaces is largely maintained

by the production of retinoic acid (RA) and TGF- β by epithelial cells. Together, these two

factors imprint local DCs to adopt a tolerogenic phenotype [14]. These DCs produce RA and

TGF- β that synergize to induce the formation of T regulatory cells (Tregs) in draining lymph

nodes [15-17]. Tregs suppress immune responses by migrating to effector sites where they

secrete TGF- β and IL-10 [16, 18, 19]. Additionally they can induce the expression of IL-27 in

DCs, generating Tr1 cells which also secrete IL-10 [20, 21]. Furthermore, large doses of

14

antigen can result in the deletion of specific T cells by apoptosis or generate a state of unresponsiveness termed anergy [22, 23]. In the steady state condition, these mechanisms ensure that mucosal tolerance and homeostasis is maintained. Therefore, in order to induce immunity a mucosal vaccine candidate must be able to overcome this disposition. However, there are currently no mucosal adjuvants licensed for human use, which precludes the development of mucosal vaccines.

Adjuvants

Adjuvants are substances that serve to potentiate the immunogenicity of vaccines. They comprise a wide variety of molecules and complex formulations that have the ability to enhance the magnitude- as well as the longevity of the immune response. By including adjuvants in vaccines the antigen dose can be dramatically lowered and the number of immunizations can be reduced. Furthermore, the choice of adjuvant can modulate the immune response in several ways. For example, a vaccine directed against an extracellular pathogen will be more dependent on a strong humoral (Th2) response as compared to intracellular organisms where a cytotoxic (Th1) response is more desirable. Skewing the immune system towards either type of response by choosing an appropriate adjuvant can be essential for the efficacy of the vaccine [24].

Based on their mode of action, adjuvants are often classified as being either delivery systems or immunostimulants. Delivery systems present antigen in a more accessible form and include aluminum salts, oil emulsions, virus like particles (VLPs), liposomes and micro- or nanoparticles. Immunostimulats on the other hand, potentiate the immune response by activating innate immunity, most often through pattern recognition receptors (PRRs).

Examples of immunostimulants are microbial components, bacterial toxins and their derivatives, endogenous danger signals such as cytokines, or molecules released as a result of tissue damage or inflammation. However this division is not mutually exclusive as many adjuvants fall into both categories [25].

The first report of mixing antigen with foreign substances to augment the activity of a vaccine was published in 1916 [26]. A majority of these early adjuvants were oil emulsions, and their discovery was soon followed by aluminum-based formulations [27]. Even today aluminum salts are still the most widely used adjuvants in human vaccines. In fact, apart from aluminum salts there are only 4 adjuvants licensed for the use in humans; MF59, AS03, AS04 and liposomes [28]. This also reflects a lack of knowledge about adjuvants and which mechanisms they use to modulate the immune response. A better understanding of how adjuvants function will provide the tools needed to rationally design better and safer adjuvants for the development of new vaccines.

Aluminum salts

Aluminum salts, commonly referred to as alum, has been used for over 80 years. The adjuvant

is employed in a number of human vaccines including vaccines against diphtheria, tetanus,

pertussis, hepatitis A and B, Haemophilus influenza type B, polio, Streptococcus pneumonia,

human papilloma virus among others [28, 29]. Alum has an excellent safety record and

generates strong humoral responses. However it is a potent inducer of Th2-skewed immunity

15 and is therefore unsuitable in vaccines against pathogens that require a Th1-driven cytotoxic T lymphocyte (CTL) response [30]. The mechanism behind the adjuvanticity of alum has been elusive, and the issue remains to be controversial. It was traditionally believed that alum increased antigen accessibility to antigen presenting cells (APCs) by forming a depot of persisting antigen [31]. However this notion has been challenged by many recent reports, including studies that have excised the injection depot without any negative effects on the immune response [32]. Moreover, alum was shown to induce the production of IL- 1β and IL- 18 in a caspase-1 dependent manner, which was linked to the release of uric acid , leading to the recruitment of monocyte derived inflammatory DCs [33, 34]. These findings have been correlated to the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome by alum, either directly or via the release of uric acid [35, 36]. However, there are conflicting reports showing that NALP3 is only essential for the activation of early innate responses but dispensable for the subsequent production of IgG antibody titers [37-39]. In an alternative model, the ability of alum to activate DCs in vivo was linked to alum-antigen complexes that bind to lipid moieties in the cell membrane leading to the up-regulation of CD86 and ICAM-1 [40]. In addition, DNA released from dying cells, may activate damage-associated molecular patterns (DAMPs) which also have been implicated in the adjuvant function of alum [41].

Thus, the immunomodulatory effects of alum are still not fully understood and are likely to be complex, involving multiple pathways.

Squalene based formulations; MF59 and AS03

MF59 (Novartis) is an oil in water emulsion based on microvesicles of squalene, a precursor to cholesterol, that can be derived from plants or from the liver of some fish species. MF59 is included in two vaccines licensed for the use in humans, a seasonal influenza vaccine and a vaccine against the H1N1 influenza strain [42]. The mechanism behind the adjuvant effect of MF59 is poorly known. It is not believed to involve depot formation since the persistence of antigen is not affected by the addition of MF59 [43]. However, MF59 was shown to induce the up-regulation a large number of genes involved in inflammation, cell migration and antigen presentation [44]. In agreement with these results, there are a number of reports demonstrating a massive influx of cells into the muscle tissue at the site of immunization, including macrophages, monocytes, DCs and neutrophils [44-47]. Antigen is then taken up by the infiltrating cells and transported to the draining lymph nodes [46, 47]. Furthermore the adjuvant effect of MF59 has been shown to be independent of NALP3- inflammasome but dependent on the adaptor protein MyD88 [48].

Similar to MF59, the AS03 (adjuvant system 03, GlaxoSmithKleine biologicals) is an oil in water emulsion based on squalene, that also contains DL- α-tocopherol, a form of vitamin E with immunomodulating activity. AS03 is included in a vaccine against H1N1 influenza [49].

The mode of action of AS03 has not been well documented; but it generates a mixed Th1/Th2

response. Similar to MF59, it causes local inflammation with the expression of numerous

chemokines and cytokines at the injection site. This was shown to facilitate the influx of

antigen loaded monocytes and DCs to the draining lymph node. The addition of DL- α-

tocopherol appears to result in an increased antigen up-take by APCs and an augmented

antibody response [50].

16

Monophosphoryl lipid A

Monophosphoryl lipid A (MPLA) is a modified form of lipopolysaccharide (LPS) from Salmonella minnesota [51]. It binds to toll-like receptor 4 (TLR-4) in a similar manner as native LPS, resulting in the activation of the transcription factor NF- κB and the production of pro-inflammatory cytokines. However, in contrast to LPS, where immunomodulation is accompanied with toxicity, MPLA retains the immunoenhancing effects of LPS with significantly reduced toxicity [52]. This has been attributed to a difference in the signaling pathway employed by the two molecules, where LPS signals via both the adaptor molecules MyD88 and Trif, but MPLA preferentially uses Trif [53]. MyD88 signaling induces a more rapid and potent pro-inflammatory response, resulting in a potentially more severe inflammation, compared to that induced by Trif. Interestingly, the generation of Il- 1β is dependent on MyD88- but independent of Trif signaling; and, hence, not induced by MPLA [54]. MPLA was first used in an experimental oil in water formulation, RIBI. In this system TLR-4 signaling was required for full adjuvant effect; however a residual adjuvant function was present, attributed to the vehicle solution itself [55].

AS04 (adjuvant system 04, GlaxoSmithKleine biological) is a combination of alum and MPLA that is licensed for the use in humans and is included in two vaccines against human papilloma virus and hepatitis B respectively. The addition of MPLA to alum generates an enhanced inflammatory response, which allows for a more balanced Th1/Th2 response as compared to when alum is used alone [56].

Cholera toxin

Vibrio cholera is the causative agent of cholera, an acute diarrheal disease responsible for thousands of deaths every year in regions with poor sanitary conditions [57]. The hallmark of cholera infections, is the massive out flux of water and electrolytes from the upper part of the small intestine, which is mediated by CT [58]. CT is a member of the AB

family of toxins, which also includes the heat labile enterotoxins (LT-1 and LT-II) from Escherichia coli, shiga toxin from Shigella dysenteriae, pertussis toxin from Bordella pertussis among others [59].

The AB

toxins are composed of an A subunit and a pentameric B subunit. The A subunit can

be further divided into the A1 and the A2 domains, linked via a disulfide bond. The A1

subunit harbors the enzymatic activity and the A2 subunit is non-covalently linked to the B

subunit. The B subunit of CT (CTB) binds to Gm1 gangliosides, present on virtually all

nucleated cells [60-63]. Upon binding to the cell, the toxin is endocytosed and delivered to the

endoplasmatic reticulum (ER) by retrograde vesicular transport via the golgi apparatus [64,

65]. In the ER the disulfide bond between the A1 and A2 subunits is reduced, the A1 subunit

is then unfolded and separated from the A2 and B subunits, a process that is facilitated by the

protein disulfide isomerase [66]. In order to be transported from the ER to the cytosol, CT

hijacks the ER-associated degradation (ERAD) pathway [67]. ERAD is a protein quality

control system that mediates the degradation of misfolded proteins by the proteasome in the

cytosol. It is still unclear how CT is brought to the cytosol and avoids ubiqutination and

subsequent degradation, this is possibly mediated via the Sec61 channel [68, 69]. In the

cytosol, the A1 subunit refolds, where it can bind to ADP-ribosylating factors (ARFs),

causing a confirmation change that greatly enhances the efficiency of the enzyme [70, 71].

17 The A1 subunit then catalyzes the transfer of an ADP-ribose moiety from nicotinamide adenine dinucleo tide (NAD) to the α subunit of the G protein Gs, causing it to lose its GTPase activity, thus becoming chronically active [72, 73] . In turn, Gsα activates adenylate cyclase which converts ATP to the second messenger cAMP [74]. cAMP has a wide range of effects in the cell. However the mechanism behind the severe fluid loss has been ascribed to the activation of the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelial cells. Activating the CFTR leads to an increased Cl

secretion, which is accompanied by the osmotic movements of water into the lumen of the gut [75].

In addition to the toxic effects induced by CT, the holotoxin is also a powerful adjuvant when admixed with- or conjugated to antigen. This ability was first demonstrated by administrating the toxin intravenously (iv) [76]. In subsequent studies, potent adjuvant activity has been reported for numerous systemic and mucosal delivery routes [77-85]. However the use of CT in human vaccines is precluded by its toxicity. As described, oral administration results in severe diarrhea. Furthermore, upon in. delivery CT can traffic to the central nervous system via olfactory nerves, causing inflammation in the brain [86-88]. This has been associated with the development of Bell’s palsy, or facial paralysis in humans. An LT-adjuvanted influenza vaccine administered in. was removed from the market due to a few cases of Bell’s palsy in vaccinated individuals [89]. Furthermore, human trials of a detoxified mutant of the LT toxin, LTK63, were recently halted for the same reason [90]. Hence, it appears that GM1-binding holotoxin-derived adjuvants should not be given in. because of the risk for neurotoxic side effects.

Although not definitely proven in experimental animals, the immunoenhancing ability of CT is believed to be mediated by the direct effects of the holotoxin on DCs [91-98]. Upon CT administration, DCs are activated, as demonstrated by their enhanced expression of co- stimulatory molecules including CD80, CD86 and CD40 as well as the major histocompatibility complex II (MHC II). This allows for efficient antigen presentation and priming of naïve T cells [85, 95, 99]. Furthermore, CT has been shown to promote the enhanced expression of CCR7 and CXCR4 on DCs which facilitates their migration into T cell areas where they can interact with cognate T cells [92, 100]. The maturation status and the expression of co-stimulatory molecules on DCs is further potentiated by the induction of IL- 1β [101]. CT is often reported to generate a Th2 dominated immune response to co- administered antigens based on the production IL-4, IL-5, IL-6 and IL-10, generating mainly IgG

, IgA and IgE antibody responses [81, 96, 99, 102, 103]. This Th2 skewing has been attributed to the down regulation of IL-12 by the inhibition of the transcription factor IRF8 [104]. However, there are numerous conflicting reports describing Th1 responses induced by CT, including IFN- γ production and the effective induction of CTLs [78-80, 105-108].

Adding to the complexity, there are also a number of recent studies demonstrating a Th17 response following CT immunization [79, 109-111].

As aforementioned, the molecular mechanisms behind the toxic effects of CT, are relatively

well established. In contrast, the underlying basis for the adjuvant effect of CT is still

incompletely understood. Site directed mutagenesis have been a useful tool in deciphering the

18

relative contribution of the enzymatic activity as well as the role of Gm1 ganglioside binding in the immunomodulating ability of CT [61]. CT mutants that lack Gm1 ganglioside binding display substantially diminished toxicity [112]. Although these constructs were not tested for adjuvant function, the importance of Gm1 ganglioside binding in the adjuvant activity of CT has been supported by studies using Gm1 ganglioside deficient mice. In these mice, immunization with CT failed to induce T cell proliferation as well as antibody responses [94].

In addition, a functional CTA1 subunit also plays an important role in mediating the full immunoenhancing effect of CT. This is evident in studies revealing a poor adjuvant effect of CTB alone and supported by elegant studies by Giuliani et al showing that mutated LT with some enzymatic activity (LTR72) were more adjuvant active than mutants with no enzymatic activity (LTK63) [80, 113] . Importantly, the CTA1-DD fusion protein (see below), which exhibits comparable adjuvant activity to CT holotoxin, provides strong evidence that the ADP-ribosylating ability of the A1-subunit induces potent immune responses [114].

Noteworthy, it is currently unclear how adjuvanticity in mutated enzymatically inactive AB

holotoxins is retained, especially in comparison to the weak adjuvant ability of the CTB or LTB molecules [82, 115, 116]. Furthermore, the requirement for cAMP production in the adjuvant effect of CT is not entirely clear. It has been suggested that other attributes of the A subunit, distinct from its enzymatic activity, such as intracellular transport or the interaction with ARFs could account for the adjuvant function in mutated enzymatically inactive AB

toxins [61, 117].

CTA1-DD

To circumvent the toxicity of CT in future human vaccines , the fusion protein CTA1-DD was developed [114]. CTA1-DD is composed of the enzymatically active A1 subunit from CT and a DD moiety from Staphylococcus aureus protein A. Contrary to the holotoxin, which binds to Gm1 gangliosides as previously described, the DD domain targets Fc- and Fab fragments on immunoglobulins, preferentially of the IgG subclasses [114, 118]. Due to the different binding properties of CTA1-DD it is completely non-toxic and safe even after in.

administration, because it cannot bind Gm1 ganglioside and, hence does not accumulated in the olfactory nerve or bulb[114, 119].

Upon immunization, CTA1-DD generates a mixed Th1/Th2 response, resulting in augmented

T cell proliferation, GC formation, antibody production as well as CTL activity [114, 120-

122]. CTA1-DD has been tested and proven to be safe using a variety of mucosal and

parenteral immunization protocols in both mice and macaques [123]. Furthermore, the fusion

protein has been used in combination with a large number of antigens and has been shown to

confer protective responses in different disease models including chlamydia, rotavirus,

influenza and Helicobacter pylori [106, 124-127]. In addition to mixing the adjuvant with

relevant antigens, CTA1-DD offers the possibility to incorporate peptide epitopes into the

fusion protein itself. The significance of this concept was demonstrated using the universal

influenza vaccine candidate, matrix protein 2 (M2e). Mice immunized in. with the CTA1-

M2e-DD vaccine were fully protected against a lethal dose of live challenge influenza virus

[126].

19 The adjuvant activity of CTA1-DD is dependent on the ADP-ribosylating A1 subunit as demonstrated by the inability of the enzymatically inactive mutant, CTA1R7K-DD, containing a single point mutation in Argenine (R) -7 to Lysine (K), to promote immune responses [118]. Interestingly, this inactivated construct promotes tolerance as opposed to immunity. By incorporating known epitopes involved in autoimmune diseases into the molecule, disease progression can be prevented or significantly ameliorated as was recently shown using a mouse model for rheumatoid arthritis [128]. This concept is currently being further evaluated for the treatment of type 1 diabetes and multiple sclerosis.

Given that CTA1-DD binds to immunoglobulins, there is the potential that the fusion protein could promote the formation of immune complexes when administered in vivo. Therefore, the involvement of immune complex formation in the adjuvant effect of CTA1-DD was assessed using mice deficient in the Fc-receptors FcγRIIb and FcεR. These mice displayed unaltered immune responses when immunized with the adjuvant, suggesting that immune complex formation was not involved in the adjuvant function of CTA1-DD [122]. Importantly, CTA1- DD appeared not to be bound to immunoglobulins in serum following injections [122].

However, it was recently documented that ex vivo generated CTA1-DD/IgG immune complexes could enhance the adjuvant function mediated by mast cells in vivo [129].

CTA1-DD was originally constructed to target B cells [114]. The fusion protein has been shown to bind both mouse and human B cells, resulting in the up-regulation of the co- stimulatory molecule CD86 [114, 130]. However, CTA1-DD can also promote immune responses independently of B cells, as demonstrated by comparable levels of T cell proliferation in mice deficient in B cells as compared to WT mice, indicating that other APCs, presumably DCs, are also activated by CTA1-DD [126]. Thus, CTA1-DD affects multiple cell types and its function is not restricted to the binding of B cells as was originally hypothesized.

The present thesis work focuses on the in vivo localization of CTA1-DD and the dependence on DCs and FDCs respectively for the adjuvant function.

The complement system

The complement system consists of a complex array of proteins found in plasma and on cell surfaces. The term “complement” originates from its ability to complement the antibacterial effects of antibodies. The effector functions of the complement system include opsonization of foreign substances, direct lysis of pathogens via the membrane attack complex (MAC) and the pro-inflammatory activities of its cleavage products, termed anaphylatoxins. Furthermore, complement receptors on B cells and FDCs are involved in enhancing B cell activation and GC reactions.

Complement activation can be initiated via three distinct pathways; the classical, lectin and

alternative pathways (Fig. 1). The classical pathway is dependent on complement fixing

antibodies bound to a foreign substance; the Fc region of bound antibodies can bind to the so

called C1 complex which generates the C3 convertase by an autocatalytic process. The C3

convertase is central to all three complement pathways, its function is to cleave C3 into C3a

(anaphylatoxin) which has a pro-inflammatory function and C3b which functions as an

20

opsonin. Moreover, the C3 convertase promotes further complement activation, and ultimately the formation of the MAC complex [131]. The lectin pathway is similar to the classical pathway, but it is initiated by mannose-binding lectin (MBL) or ficolins rather than antibodies. MBL and ficolins can bind to carbohydrates on pathogenic surfaces, forming a complex with MBL-associated serine proteases which are analogous to the C1 complex of the classical pathway [132, 133].

In contrast to the classical- and lectin pathways, the alternative pathway is initiated by spontaneous hydrolysis of C3, occurring mainly on the surface of microorganisms. Together with factor B and factor D, cleaved C3 forms the C3 convertase, generating further C3 deposition in an analogous way to the classical- and lectin pathways [134].

Further deposition of cleaved C3 in association with C3 convertases, forms the C5 convertase, which constitutes the basis for the formation of the MAC complex. The MAC complex induces cell lysis of pathogens by forming pores in the cell membrane [135].

To prevent inappropriate complement activation on endogenous cells there are a number of factors regulating the complement cascade. These components include factor I and the decay- accelerating factor (DAF) which acts to inactivate bound C3- and C4 cleavage products or C3 convertases, respectively. In order to prevent inactivation of complement bound to foreign substances, these proteins require cofactors that are only expressed on host cells. These cofactors include CD46, complement receptor 1, C4 binding protein and factor H [136].

Components of activated complement are recognized by a variety of receptors. The anaphylatoxins bind G-protein-coupled receptors primarily expressed on granulocytes, macrophages, monocytes, neutrophils, mast cells and DCs corresponding to the pro- inflammatory properties of these molecules [137]. Opsonizing proteins are recognized by the complement receptors 1-4 and CRig. Complement receptor 1 (CR1/CD35) and 2 (CR2/CD21) are splice forms of the Cr2 gene [138]. The former is expressed on erythrocytes, monocytes, neutrophils and B cells whereas the latter is found mainly on B cells and FDCs. CR1 and CR2 are important in the transport- and deposition of immune complexes on FDCs, a process which will be further discussed in the section describing GCs. CR2 is also included in a co- receptor complex with the BCR (B cell receptor), together with CD19 and CD81 (TAPA-1).

This co-receptor substantially reduces the threshold for activation of naïve B cells,

significantly improving the ability of B cells to respond to low affinity antigens [139]. CR3

and CR4 are heterodimers composed of CD11b/CD18 and CD11c/CD18, respectively. They

are mediating phagocytosis of complement coated pathogens, CR3 is mainly expressed on

monocytes, neutrophils and NK cells whereas CR4 is primarily found on macrophages [140,

141]. CRIg is involved in clearing complement opsonized substances and is expressed on

Kupffer cells in the liver [142]. Together the complement system has a wide range of effects

and is involved in both innate- and adaptive immune functions.

21

The cell types and the microanatomy of the spleen

The primary function of the spleen is to filter blood in order to launch a rapid response against blood borne pathogens or to remove debris such as old and dying erythrocytes. It is also the main inductive site for immune responses initiated using the intraperitoneal- or intravenous immunization routes. Blood enters the spleen via the splenic artery which branch into several central arterioles. The central arterioles branches further to smaller vessels that empties directly into the red pulp or into the marginal sinus. The blood is then filtered thru the marginal zone (MZ) and further into the red pulp where it passes into venous sinuses that collect into the efferent vein, vena linealis. There are three functionally and phenotypically

Figure 1. Schematic overview of the complement system.

22

distinct compartments of the spleen, the red pulp, the white pulp and the MZ [143]. The most abundant cell type in the red pulp are the red pulp macrophages; they have an important function in phagocytizing erythrocytes and iron recycling [144]. Other cell types found in the red pulp include DCs, natural killer cells, plasma cells as well as a small number of B- and T cells.

The white pulp host B cell follicles as well as organized T cell areas (also referred to as the periarteriolar lymphoid sheets, PALS). The B cell follicles comprise a large population of follicular B cells as well as a small number of FDCs and follicular stromal cells. The latter two cell types secrete the chemokine CXCL13, which attracts B cells via stimulation of their CXCR5 receptors [145]. During an immune response GCs can form in the center of B cell follicles, giving rise to high affinity memory B cells as well as antibody secreting plasma cells. Moreover, tingible body macrophages found in the GC remove apoptotic cells generated during the GC reaction [146]. The T cell areas contain CD4

and CD8

T cells and DCs.

CCL19 and CCL21 are secreted by stromal cells in the T cell area, thereby recruiting CCR7 expressing T cells [147]. MOMA-2

macrophages are present in both B cell follicles and T cell areas, although incompletely studied, they have been shown to be important in providing a local source of complement C3 [148].

The MZ is located to the interface between the white pulp and the red pulp, it contains the marginal sinus which allows for a close contact between the slowly percolating blood and cells in the MZ. The cells of the MZ are unique to this compartment and have specialized functions which allow them to rapidly respond to- and control blood borne pathogens. These cell types include two macrophage subtypes, the marginal metallophilic macrophages (MMM) and the marginal zone macrophages (MZM), marginal zone B cells (MZB) and DCs [149].

Marginal zone B cells

MZB cells constitute a population of non-recirculating cells, phenotypically and functionally distinct from follicular B cells (FOB). Naïve MZB cells differ from FOB cells in that they are IgM

, CD21

, CD23

and CD1d

, whereas FOB cells can be identified as being IgD

, CD21

, CD23

and CD1d

[150]. The relatively high expression of CR2 on MZB cells allows them to bind and transport immune complexes into the follicle; this function is important in GC formation and will be further discussed in the section describing antigen localization to FDCs. The MHC 1-like molecule CD1d, expressed on MZB cells, enables the presentation of lipid antigens to invariant natural killer (iNKT) cells and has been implicated in the production of anti-lipid antibodies by MZB cells [151, 152]. In addition, MZB cells have been shown to be involved in the formation of antibodies towards T cell independent (TI) antigens, mice that lack MZB cells display reduced IgM, IgG

and IgG

antibody titers to TI antigens, while titers against T cell dependent (TD) antigens remained unaffected [153].

These responses are dominated by extrafolliclular plasma cells and occur within a few days

following immunization [154, 155]. This unique ability of the MZB cells to launch rapid

responses has been attributed to their low threshold of activation as compared to FOB cells

[156]. However, the activity of MZB cells is not restricted to TI antigens, they can also

respond to TD antigens and participate in GC reactions where they undergo somatic

hypermutation and class switching [157, 158].

23 Macrophages of the marginal zone

There are two distinct populations of macrophages in the MZ, the marginal zone macrophages (MZM) and the marginal metallophilic macrophages (MMM). They are distinguished by their differential expression of surface markers as well as by their location in the MZ. MZM are found in the outer MZ whereas MMM are found on the inner side, in close contact with the marginal sinus, bordering the white pulp [149]. MMM are identified by the antibody MOMA- 1 which recognizes sialoadhesin (CD169, siglec-1) [159, 160]. Sialoadhesin is a receptor for sialylated bacteria and has been shown to mediate the phagocytosis of sialylated strains of Neisseria meningitides [161]. MZM can be identified by the antibody ERTR-9 which recognizes the specific intracellular adhesion molecule-grabbing nonintegrin receptor 1 (SIGN-R1). SIGN-R1 binds to polysaccharides of capsulated bacteria and has been shown to facilitate the uptake of Streptococcus pneumoniae [162-164]. Another marker for MZM is the macrophage receptor with collagenous structure (MARCO), a scavenger receptor which binds a range of bacterial products including LPS, thereby mediating the phagocytosis of blood borne bacteria by MZM [165-167]. Furthermore MZM express the closely related scavenger receptor A (SR-A), which also is an important phagocytic receptor, recognizing various bacterial products [168-172].

Both MZM and MMM are highly phagocytic and have been shown to trap a wide range of particulate- and non-particulate antigens [162, 164, 165, 173-182]. This ability is crucial in preventing the hematogenic spread of blood borne infections as demonstrated by the reduced pathogen clearance and diminished survival of systemically infected mice lacking these macrophage subsets [162, 173, 174, 180, 182]. With the exception of some reports where the specific role of the receptors SIGN-R1 or MARCO have been investigated [162, 165, 180], many of the studies have used clodronate liposomes to deplete MZM and MMM [173, 174], or alternatively employed osteopetrotic (op/op) mice, which correspondingly lacks both subsets [182]. Thus, it is difficult to distinguish between the respective roles of MZM or MMM in pathogen clearance in the systems that have been studied.

Although essential for removing disseminated pathogens from the blood, MZM and MMM do not appear to have a role in priming T cells since T cell priming was intact in mice deficient in both MZM and MMM populations [173, 174, 182]. This notion is also supported by their apparent lack of MHC II molecules [183]. However, MZM and MMM may have other roles in the induction of immune responses. For example, they have been shown to be the major producers of type 1 interferons following systemic injection with Herpes simplex virus [183].

Furthermore, MMM have been implicated in the transfer of antigens to CD8

DC, enabling cross presentation and CTL induction [184].

Dendritic cells

In the spleen, classical DCs are found in the red pulp, the T cell areas and in the MZ. They

can be divided into three distinct subtypes based on their function as well as their expression

of surface molecules; CD8a

CD11b

DEC205

DCs, CD8α

CD11b

DEC205

CD4

and CD8α

CD11b

DEC205

CD4

DCs, here I will refer to the former as CD8α

DCs and the latter two

as CD8α

DCs [185-187] . CD8α

DCs are primarily located to the splenic bridging channels

24

and in the red pulp, whereas CD8α

DCs are found in the T cell areas [188, 189]. However, recently a CD8α

DC population was described to reside in the MZ and identified by the langerin (CD207) marker [190].

CD8α

and CD8α

DCs differ in their ability to present antigen to T cells. CD8α

, but not CD8α

DCs can take up apoptotic cells and are capable of cross presenting exogenous cell associated- as well as soluble antigen on MHC class I molecules [189, 191-193]. This correlates with the superior ability of CD8α

DCs to prime MHC class I restricted CD8 T cells. Conversely, CD8α

DCs more efficiently induces proliferation of CD4 T cells that recognizes peptides presented on MHC class II. This dichotomy has been demonstrated in vitro , using either CD8α

or CD8α

DCs as APCs in culture [193], or in vivo by targeting antigen to the respective DC population [189] or by using knockout mice lacking the CD8α

DC population [194]. In addition to their divergent capacity with regard to APC function, CD8α

and CD8α

DCs also differ in their respective cytokine secretion profiles. CD8 α

DCs are able to secrete IL-12 upon activation, inducing Th1 T cells and IFN- γ production, whereas CD8α

DCs elicit a more Th2 skewed response, mainly resulting in the secretion of IL-4 and IL-10 [195-197].

The induction of polarized T cell responses is not only determined by the intrinsic differences between the different DC subtypes. In fact there is a significant plasticity within the DC population to modulate the immune responses depending on the various activation stimuli.

DCs express a wide array of PRRs, including TLRs, NOD-like receptors (NLRs), C-type lectin receptors etc. [198]. Different microbial products bind to different receptors and therefore influence the cytokine pattern released by DCs and consequently the polarization of T cell responses. For example LPS from Escherichia coli signals via TLR4, promoting the secretion of IL-12 which drives Th1 induction. In contrast LPS from a different bacterium, Porphiromonas gengivalis signals via TLR2 which generates Th2 type of responses [199].

Together, these intrinsic and extrinsic signals govern the polarizing activities by DCs, enabling the induction of tailored responses specifically aimed at combating different types of pathogens.

DCs of the spleen are resident, non-migratory cells that arrive as precursors via the blood

stream [200]. Under steady state conditions the majority of splenic DCs are immature, that is

they express low levels of MHC- and T cell co-stimulatory molecules and produce low

amounts of cytokines [201-203]. However upon exposure to microbial products they can be

induced to mature, up-regulating their levels of MHC- and co-stimulatory molecules, i.e

CD40, CD80 and CD86 [186, 188].This activation correlates with the expression of CCR7

which facilitates the migration into T cell areas where they can prime naïve T cells [204]. In

contrast to the spleen, lymph nodes also harbor a population of migratory DCs in addition to

the resident population. The migratory DCs comprise a heterogeneous population of mature

DCs that continuously travel from the periphery to draining lymph nodes [205-207]. In lymph

nodes they can present peripheral antigen directly to T cells, or alternatively transfer antigens

to resident DCs which in turn can prime naïve T cells [208, 209].

25

T cell differentiation

Following CD4 T cell priming activated T cells can differentiate into distinct subsets that are distinguished by their diverse effector functions and cytokine secretion patterns. The induction of these subpopulations is influenced by the local cytokine milieu at the priming event [210]. Central to this process is the activation of the signaling transducer and activator of transcription (STATs) proteins, these transcription factors are induced by signaling thru cytokine receptors and in turn regulate the expression of the different master transcription factors that define subset differentiation. Up until relatively recently only the Th1 and Th2 subsets had been described, but today we know that several additional lineages exist including the Th17-, Treg and T follicular helper (Tfh) cell subsets [211]. An overview of the different CD4 T cell subsets, the cytokines involved in their differentiation and their secretion profiles is presentment in (Fig. 2).

Th1

Th1 cells are primarily involved in the protection against viruses and intracellular bacteria;

their effector functions include the activation of macrophages as well as the expansion of cytolytic CD8 T cells. IFN- γ is the signature cytokine produced by Th1 cells and it is also involved in the initial induction of Th1 differentiation. IFN- γ activates STAT1, which promotes the expression of the Th1 master regulator, T-bet. T-bet drives transcription of the

Figure 2. CD4 T cell differentiation.

26

IFN- γ gene; this creates a positive feedback loop which amplifies the Th1 response [212-214].

T-bet also functions to negatively regulate the Th2 transcription factor GATA-3, which further reinforces the Th1 lineage commitment [215]. Additionally, T-bet mediates the up- regulation of the IL-12 receptor on the T cell [213]. IL-12 is involved in a second signaling pathway that drives Th1 differentiation, this pathway is dependent on the induction of STAT4 which promotes the secretion of IFN- γ thus augmenting Th1 development [216].

Consequently, mice deficient in IL-12, IL-12R, T-bet or STAT4 exhibit severely diminished Th1 responses [216-219].

In addition to IL-12 and IFN- γ, other factors can also influence Th1 differentiation. For example, IL-18 and IL-27 synergizes with IL-12 to drive Th1 induction, this is illustrated by the impaired ability to form Th1 responses in mice deficient in IL-18 or IL-27 [220-223].

Furthermore Notch receptors and their ligands have been implicated in Th1 development.

Notch 1 and Notch 2 are activated by the ligands Delta-like (Dll) 4 and 1 respectively [224].

Their involvement in Th1 differentiation has been demonstrated by inhibiting Notch signaling, resulting in diminished Th1 responses [225] or by inducing the expression of Dll4 or Dll1 on DCs which promotes Th1 development [226, 227]. Interestingly, the expression of Dll4 on CD8α

, but not CD8α

DCs, has been shown to be induced by LPS, allowing for IL- 12 independent Th1 differentiation [228].

Th2

Th2 cells are generated in response to extracellular parasites such as helminthes or nematodes.

A hallmark of Th2 differentiation is the secretion of IL-4, IL-5 and IL-13, which ultimately

results in the recruitment of mast cells and eosinophils and promotes immunoglobulin isotype

switching from IgM to IgG

and/or IgE. Differentiation of Th2 cells can be induced by IL-4,

which activates STAT6 which in turn induces the expression of GATA-3, the master

transcription factor for Th2 development [229, 230]. GATA-3 promotes the secretion of IL-4

from the Th2 cells which drives further Th2 differentiation. The source of the IL-4

responsible for early STAT6 activation remains unclear. Basophils are known to secrete IL-4

and are therefore likely to contribute to Th2 development, however there is much controversy

regarding the extent of their contribution [231]. Under some conditions, in vivo Th2

development can occur in the absence of STAT6, suggesting that signals other than IL-4 are

able to drive Th2 differentiation [232, 233]. A second model for Th2 induction revolves

around the notion that weak TCR (T cell receptor) signaling generally favors Th2- as opposed

to Th1 development. This has been demonstrated using peptides that interact with the TCR

with low affinity or by using peptides of low concentration [234, 235]. Under these

circumstances TCR signaling causes a slight increase in GATA-3 expression and

simultaneously induces the production of IL-2. IL-2 activates STAT5 which synergizes with

GATA-3 to produce IL-4 which further drives Th2 differentiation [235]. In contrast, strong

TCR signaling reduces IL-2 expression and GATA-3 expression [235]. Additionally the notch

ligand Jagged 1 and its receptors Notch 1 and 2 are essential in the development of Th2

responses [226]. This is mediated by GATA-3 expression which is induced upon Notch

signaling [236].

27 Th17

Th17 responses are important in protection against extracellular pathogens such as bacteria or fungi. Th17 development is initiated by the combination of TGF- β and IL-6 [237]. TGF-β induces the expression of RORγt, the Th17 master regulator, which synergizes with RORα to promote Th17 development [238, 239]. TGF- β also induces the expression of Foxp3, the transcription factor involved in Treg development [240]. The balance between the induction of inflammatory Th17 cells or anti-inflammatory Tregs depends on the presence or absence of IL-6 [241]. IL- 6 is a potent inducer of STAT3 which drives the expression of RORγt, thus promoting the preferential development of Th17 cells as opposed to Tregs [242]. STAT3 also induces the secretion of IL-21 which acts in an autocrine manner to potentiate Th17 development by further activation of STAT3 and RORγt [242]. Furthermore both STAT3 and RORγt up-regulates the IL-23 receptor on Th17 cells, IL-23 is important in sustaining Th17 differentiation by promoting STAT3 activation [242].

Th17 cells produce the signature cytokines IL-17A/IL-17F and IL-22. IL-17 is a pro- inflammatory cytokine that recruits neutrophils and macrophages to the inflamed tissues, whereas mice lacking IL-17 or the IL-17 receptor display an increased susceptibility to a number of infectious diseases [243]. IL-22 has a wide range of effects, it can both regulate- and induce inflammation, it also has an important protective role by improving barrier functions at mucosal sites [244-246].

T regulatory cells

To maintain homeostasis immune responses must be controlled, this is critical in order to dampen inflammation following an infection or to induce tolerance to self- or environmental antigens. This is largely accommodated by the activities of regulatory T cells (Tregs). Tregs can be divided into two main categories, those that are derived from the thymus, termed natural Tregs (nTreg) and those that are formed in the periphery termed inducible Tregs (iTreg). Both nTregs and iTregs express the transcription factor Foxp3 [247, 248]. nTregs are believed to be derived from T cells that express TCRs with high affinity for self-antigens [249, 250]. iTregs on the other hand are induced by tolerogenic factors in the periphery, one such factor, TGF- β induces the expression of Foxp3 which is required for Treg suppressor function and maintenance [240]. There is also a subset of Foxp3

iTregs, termed Tr1 cells, they are induced by IL-10, and secretion of IL-10 is also their major effector function [251, 252]. The mechanisms by which Tregs regulate immune responses are diverse and rely on both the secretion of cytokines as well as cell-contact dependent suppression. Secreted inhibitory cytokines include IL-10, TGF- β and IL-35 [19, 253, 254], in addition Tregs express the cytotoxic T lymphocyte antigen-4 (CTLA-4) which can down-modulate the stimulatory capacity of DCs [255, 256], furthermore, Tregs can mediate suppression by direct killing of target cells via Granzyme B induced cytolysis [257].

Additional Th subsets

In addition to the T cell subtypes described above, two additional lineages have recently been

defined, the Tfh- and the Th9 subset. Tfh cells are essential in orchestrating GC responses and

will be further discussed in the next section. Th9 cells can be induced by IL-4 and TGF- β,

28

they secrete the cytokine IL-9 and are involved in the protection against intestinal parasites [258-260]. Whether the Th9 cells should be considered a separate subset, or if they are, in fact, a subpopulation of Th2 cells, is a matter of debate. However, recently the transcription factor PU.1 was shown to be required for Th9 induction, suppressing GATA-3[258, 261].

Germinal centers

Within a few hours after being antigen-activated via the BCR, B cells migrate to the B/T cell zone border where they receive essential co-stimulatory signals from cognate T helper cells [262]. Migration to the border area is facilitated by an altered receptor expression; CCR7, the receptor for the chemokines CCL19 and CCL21, produced in the T cell zone, is up-regulated [263]. Furthermore the expression of the Epstein-Barr virus-induced G-protein coupled receptor 2 (EBI2) which directs B cells towards the peripheral regions of the B cell follicle is also increased [264]. B cells are prevented from migrating further towards the T cell zone by their maintained expression of CXCR5, enabling them to respond to the chemokine CXCL13, which is produced in B cell follicles. In parallel to the positioning of activated B cells, cognate T cells migrate to the B/T cell border in a similar manner, they down-regulate their expression of CCR7 and subsequently up-regulate CXCR5 [265, 266]. After having received T cell help, B cells can adopt one of two fates; they can either become short lived extrafollicular plasma cells or participate in a GC reaction [267]. Extrafollicular plasma cells provide an early wave of low affinity antibodies that are important in protection during the initial stages of an infection prior to the formation of antibodies with higher affinity. These cells down-regulate their expression of CXCR5 and CCR7 and up-regulate CXCR4, which facilitates their positioning to the bridging channels and red pulp of the spleen or in the medulla of lymph nodes [268]. In contrast, the pre-GC B cells migrate into the B cell follicles, as a consequence of down-modulating their expression of CCR7 and EBI2, hence the GC reaction can develop a few days after initial antigen encounter [264].

GCs are specialized regions of rapid cell division that form in response to infection or immunization within the B cell follicles of secondary lymphoid organs. Their primary purpose is to generate plasma cells and memory B cells of high affinity. Structurally, GCs can be further divided into two distinct regions, the light zone (LZ) and the dark zone (DZ), originally based on their histological appearance [269]. B cells of the LZ are termed centrocytes and those residing in the DZ are called centroblasts. The distinction between the LZ and DZ is maintained by the expression of CXCR5 and CXCR4. CXCR4

centroblasts are attracted to the DZ by the chemokine CXCL12 which is expressed at higher levels in the DZ as compared to the LZ, whereas CXCR4

centrocytes migrate towards CXCL13 which is more abundantly expressed in the LZ [270].

The purpose of the compartmentalization between the LZ and the DZ was originally proposed

by MacLennan in a classical model [271]. This model describes how the selection- and

generation of high affinity B cells in the GC is accomplished; rapidly dividing centroblasts in

the DZ generate random point mutations in their Ig variable (V-) regions by somatic

hypermutation (SHM). This process produces cells that express BCRs of variable affinity for

a given antigen. Following a number of consecutive rounds of division, the centroblasts then